Hi Simon,
It appears that one of my regular unsorted SAM files is truncated.
Though the sort works fine and samtools view does not detect
truncation
in my file.sam.bam, I still get error messages after attempting to
generate
counts files.
samtools view -h file.sam.bam | python dexseq_count.py file.gff -
file.counts
Do you think that the truncation may be behind the problems?
I can't check the structure of my SAM.BAM file because its all
symbols,
though when I use samtools view, the contents runs across the screen,
and I
am unable to pinpoint any irregularity.
Thanks,
Margaret
[[alternative HTML version deleted]]
It would be helpful if you reported the actual error messages.
____________________________________________
Devon Ryan, Ph.D.
Email: dpryan at dpryan.com
Tel: +49 (0)178 298-6067
Molecular and Cellular Cognition Lab
German Centre for Neurodegenerative Diseases (DZNE)
Ludwig-Erhard-Allee 2
53175 Bonn, Germany
On Oct 6, 2013, at 10:51 PM, Margaret Linan wrote:
> Hi Simon,
>
> It appears that one of my regular unsorted SAM files is truncated.
>
> Though the sort works fine and samtools view does not detect
truncation
> in my file.sam.bam, I still get error messages after attempting to
generate
> counts files.
>
> samtools view -h file.sam.bam | python dexseq_count.py file.gff -
> file.counts
>
> Do you think that the truncation may be behind the problems?
>
> I can't check the structure of my SAM.BAM file because its all
symbols,
> though when I use samtools view, the contents runs across the
screen, and I
> am unable to pinpoint any irregularity.
>
> Thanks,
> Margaret
>
> [[alternative HTML version deleted]]
>
> _______________________________________________
> Bioconductor mailing list
> Bioconductor at r-project.org
> https://stat.ethz.ch/mailman/listinfo/bioconductor
> Search the archives:
http://news.gmane.org/gmane.science.biology.informatics.conductor
After properly sorting, using Simons method and then using the dexseq
count
program I get error messages
These are a sample of the error messages:
UserWarning: Read HWI-ST522:121:D0V1CACXX:8:1106:20332:74088 claims to
have
an aligned mate which could not be found. (Is the SAM file properly
sorted?)
"which could not be found. (Is the SAM file properly sorted?)" )
/packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598:
UserWarning: Read HWI-ST522:121:D0V1CACXX:8:1106:21152:96414 claims to
have
an aligned mate which could not be found. (Is the SAM file properly
sorted?)
"which could not be found. (Is the SAM file properly sorted?)" )
/packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598:
UserWarning: Read HWI-ST522:121:D0V1CACXX:8:1106:2563:90289 claims to
have
an aligned mate which could not be found. (Is the SAM file properly
sorted?)
"which could not be found. (Is the SAM file properly sorted?)" )
/packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598:
UserWarning: Read HWI-ST522:121:D0V1CACXX:8:1106:2842:88054 claims to
have
an aligned mate which could not be found. (Is the SAM file properly
sorted?)
"which could not be found. (Is the SAM file properly sorted?)" )
/packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598:
UserWarning: Read HWI-ST522:121:D0V1CACXX:8:1106:6895:17883 claims to
have
an aligned mate which could not be found. (Is the SAM file properly
sorted?)
"which could not be found. (Is the SAM file properly sorted?)" )
/packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598:
UserWarning: Read HWI-ST522:121:D0V1CACXX:8:1106:7069:24372 claims to
have
an aligned mate which could not be found. (Is the SAM file properly
sorted?)
"which could not be found. (Is the SAM file properly sorted?)" )
Thanks,
Margaret
On Sun, Oct 6, 2013 at 2:29 PM, Devon Ryan <dpryan@dpryan.com> wrote:
> It would be helpful if you reported the actual error messages.
>
> ____________________________________________
> Devon Ryan, Ph.D.
> Email: dpryan@dpryan.com
> Tel: +49 (0)178 298-6067
> Molecular and Cellular Cognition Lab
> German Centre for Neurodegenerative Diseases (DZNE)
> Ludwig-Erhard-Allee 2
> 53175 Bonn, Germany
>
> On Oct 6, 2013, at 10:51 PM, Margaret Linan wrote:
>
> > Hi Simon,
> >
> > It appears that one of my regular unsorted SAM files is truncated.
> >
> > Though the sort works fine and samtools view does not detect
truncation
> > in my file.sam.bam, I still get error messages after attempting to
> generate
> > counts files.
> >
> > samtools view -h file.sam.bam | python dexseq_count.py file.gff -
> > file.counts
> >
> > Do you think that the truncation may be behind the problems?
> >
> > I can't check the structure of my SAM.BAM file because its all
symbols,
> > though when I use samtools view, the contents runs across the
screen,
> and I
> > am unable to pinpoint any irregularity.
> >
> > Thanks,
> > Margaret
> >
> > [[alternative HTML version deleted]]
> >
> > _______________________________________________
> > Bioconductor mailing list
> > Bioconductor@r-project.org
> > https://stat.ethz.ch/mailman/listinfo/bioconductor
> > Search the archives:
> http://news.gmane.org/gmane.science.biology.informatics.conductor
>
>
[[alternative HTML version deleted]]
That's a warning, not an error and you can likely ignore it. As Simon
alluded in his earlier reply, the script expects mates in a pair to
follow one another. When they don't, the warning you observed is
issued. With tophat, this can occur when (1) you don't specify --no-
mixed or (2) you specify --fusion-search. Tophat is known to not
always produce proper flags.
Regards,
Devon
____________________________________________
Devon Ryan, Ph.D.
Email: dpryan at dpryan.com
Tel: +49 (0)178 298-6067
Molecular and Cellular Cognition Lab
German Centre for Neurodegenerative Diseases (DZNE)
Ludwig-Erhard-Allee 2
53175 Bonn, Germany
On Oct 7, 2013, at 12:21 AM, Margaret Linan wrote:
> After properly sorting, using Simons method and then using the
dexseq count program I get error messages
>
> These are a sample of the error messages:
>
> UserWarning: Read HWI-ST522:121:D0V1CACXX:8:1106:20332:74088 claims
to have an aligned mate which could not be found. (Is the SAM file
properly sorted?)
> "which could not be found. (Is the SAM file properly sorted?)" )
> /packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598: UserWarning: Read HWI-
ST522:121:D0V1CACXX:8:1106:21152:96414 claims to have an aligned mate
which could not be found. (Is the SAM file properly sorted?)
> "which could not be found. (Is the SAM file properly sorted?)" )
> /packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598: UserWarning: Read HWI-
ST522:121:D0V1CACXX:8:1106:2563:90289 claims to have an aligned mate
which could not be found. (Is the SAM file properly sorted?)
> "which could not be found. (Is the SAM file properly sorted?)" )
> /packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598: UserWarning: Read HWI-
ST522:121:D0V1CACXX:8:1106:2842:88054 claims to have an aligned mate
which could not be found. (Is the SAM file properly sorted?)
> "which could not be found. (Is the SAM file properly sorted?)" )
> /packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598: UserWarning: Read HWI-
ST522:121:D0V1CACXX:8:1106:6895:17883 claims to have an aligned mate
which could not be found. (Is the SAM file properly sorted?)
> "which could not be found. (Is the SAM file properly sorted?)" )
> /packages/python/python-2.7.3/lib/python2.7/site-
packages/HTSeq/__init__.py:598: UserWarning: Read HWI-
ST522:121:D0V1CACXX:8:1106:7069:24372 claims to have an aligned mate
which could not be found. (Is the SAM file properly sorted?)
> "which could not be found. (Is the SAM file properly sorted?)" )
> Thanks,
> Margaret
>
>
> On Sun, Oct 6, 2013 at 2:29 PM, Devon Ryan <dpryan at="" dpryan.com="">
wrote:
> It would be helpful if you reported the actual error messages.
>
> ____________________________________________
> Devon Ryan, Ph.D.
> Email: dpryan at dpryan.com
> Tel: +49 (0)178 298-6067
> Molecular and Cellular Cognition Lab
> German Centre for Neurodegenerative Diseases (DZNE)
> Ludwig-Erhard-Allee 2
> 53175 Bonn, Germany
>
> On Oct 6, 2013, at 10:51 PM, Margaret Linan wrote:
>
> > Hi Simon,
> >
> > It appears that one of my regular unsorted SAM files is truncated.
> >
> > Though the sort works fine and samtools view does not detect
truncation
> > in my file.sam.bam, I still get error messages after attempting to
generate
> > counts files.
> >
> > samtools view -h file.sam.bam | python dexseq_count.py file.gff -
> > file.counts
> >
> > Do you think that the truncation may be behind the problems?
> >
> > I can't check the structure of my SAM.BAM file because its all
symbols,
> > though when I use samtools view, the contents runs across the
screen, and I
> > am unable to pinpoint any irregularity.
> >
> > Thanks,
> > Margaret
> >
> > [[alternative HTML version deleted]]
> >
> > _______________________________________________
> > Bioconductor mailing list
> > Bioconductor at r-project.org
> > https://stat.ethz.ch/mailman/listinfo/bioconductor
> > Search the archives:
http://news.gmane.org/gmane.science.biology.informatics.conductor
>
>
Hi Margaret
On 06/10/13 22:51, Margaret Linan wrote:
> It appears that one of my regular unsorted SAM files is truncated.
If the SAM file is unsorted, this might be annoying but okay. If it is
sorted by position, you have a problem: All genes on chromosomes with
high numbers will be missing and hence wrongly appear downregulated in
this sample.
> Though the sort works fine and samtools view does not detect
truncation
> in my file.sam.bam, I still get error messages after attempting to
generate
> counts files.
>
> samtools view -h file.sam.bam | python dexseq_count.py file.gff -
> file.counts
>
> Do you think that the truncation may be behind the problems?
Sure, if the truncation causes some mates to be missing, htseq-count
will complain about it. Whether you need to worry about it depends on
whether it affects only a small fraction of the reads or more.
> I can't check the structure of my SAM.BAM file because its all
symbols,
> though when I use samtools view, the contents runs across the
screen, and I
> am unable to pinpoint any irregularity.
You can write the bam file back into a sam file ("samtools view
abc_sorted.bam > abc_sorted.sam") to inspect it.
Simon
